The application is the first competitive review of the PI's MERIT award, during which much progress was made on gene silencing in Saccharomyces cerevisise. During this period, the genetic approaches that have been a strength of the lab have been complemented with new expertise in protein biochemistry, protein structure, genomics and computation. The three big goals of this research are: to understand how RNAi-independent gene silencing occurs leading to the formation of heterochromatin, how specialized structures of chromatin achieve their epigenetic inheritance, and how nutrition can affect epigenetic processes. On the mechanism of silencing, the PI proposes experiments to resolve the contradictory data from the Gross and Widom labs regarding how Sir proteins limit transcription factors' functions in heterochromatin. The NAD metabolite 2-0- Acetyl ADP Ribose, the product of the Sir2 histone deacetylase has gained much interest from in vitro suggestions for a role in silencing. The PI proposes three possible roles for the metabolite and critical in vivo tests of each role. Work on comparative silencing in related species has identified a Sir3 backup mechanism and provide a context that will allow testing of the growth versus oozing models for how heterochromatin spreads. Computational analysis of silencer sequences revealed a Rap1 binding site that is more conserved between species than are Rap1 binding sites within the same species. A hypothesis for this unprecedented conservation and a compelling test are proposed. With respect to the inheritance of the silenced state, a model is presented involving the segregation of Sir protein complexes on histone H3-H4 tetramers that makes specific quantitative predictions about how the stability of these silenced states should vary with the length of the silenced domain. An elegant test of the hypothesis is described that measures rates of change in heritable transcription states. In the recent past, the PI has uncovered links between H2A.Z acetylation, boundary function and the Bdf1 and Yta7 bromodomain proteins, which motivate experiments to uncover how chromatin boundaries function. Isotype-specific modifications and functions of the two H2Bs of yeast will be explored. Finally, the PI has uncovered a fascinating link between nutrition, in the context of folic acid, and heritable gene silencing through histone hypomethylation, whose mechanistic basis will be tested. PUBLIC HEALTH RELEVANCE: The inheritance of stable states of gene expression is relevant to the orderly development of all plants and animals and to the propagation of disease states such as cancer. Moreover, the link between nutritional status and epigenetic states is particularly important because 9% of people have two defective copies of a gene that, based upon the results here, are likely to impact heritable features on their chromosomes.